Fig. 2

Generation of affinity-improved mutants of CBTAU-27.1 and CBTAU-28.1

a Structure-based design of mutants around Pro³¹² (left panel) and Val³¹³ (right panel). The tau epitope is illustrated as in Fig. 1b. Antibody loops and the key residues interacting with Pro³¹² and Thr⁹⁴ are plotted in white. Proposed mutations are shown as orange sticks on top of the corresponding wild-type side chains. Ser^27D is mutated to tyrosine (left panel) to enlarge the hydrophobic pocket of Pro³¹², and Thr⁹⁴ is mutated to isoleucine (right panel) to fill the empty cavity surrounding Val³¹³ and Leu³¹⁵. By introducing both mutations, additional hydrophobic contacts between tau and the antibody loops could be formed, potentially resulting in a lower desolvation penalty and increased affinity. b Schematic representation of the CBTAU-28.1 affinity maturation process by random mutagenesis. Mutations were introduced randomly by error prone PCR in the coding sequence for the single-chain variable fragment (scFv) directed against the CBTAU-28.1 epitope. M13 phage libraries displaying the scFv were screened against rtau and peptide A6940. Affinity-matured variants were identified by phage ELISA and converted into an IgG1 format to assess binding in solution. c and d Association and dissociation profiles for parental and affinity improved CBTAU-27.1 (c) and CBTAU-28.1 (d) variants to their corresponding cognate peptides as determined by Octet biolayer interferometry. Affinities as determined by ITC (K_d) are shown on the individual graphs. (e and f) Co-crystal structures of the Fabs of dmCBTAU-27.1 (e) and dmCBTAU-28.1 (f) with tau peptides A8119 and A7731, respectively. Antibodies are illustrated as molecular surfaces (colored as in panel A), together with tau epitopes as sticks with yellow carbons. The corresponding parental co-crystal structures have been aligned using their variable regions, and their tau epitopes are shown as blue mesh on top of the mutant epitopes.